1. Field of the Invention
The present invention relates to purified and isolated antigens for the detection of bovine or swine cysticercosis and methods of synthesis of recombinant antigens using genetic engineering techniques.
2. Description of the Art
Cysticercosis is a parasitic disease of cattle and swine caused by the larval (cyst) stage of the tapeworm Taenia saginata in cattle and Taenia solium in swine. Ingestion by man of viable larvae in undercooked meat results in intestinal tapeworm infection (taeniasis). The meat of these animals contains larvae that mature, in the intestinal tract, into adults that burrow into the mucosa and at the same time attach themselves to the bowel wall with scolices which bear hooks. The mature worm then progressively develops to lengths of 3 to 6 meters (10 to 20 feet). Clinical manifestations of the intestinal infection arise from the physical mass of the worms, the trauma to the intestinal mucosa, reaction to worm metabolites, and competitive uptake of nutrition. Of particular concern is taeniasis caused by the swine tapeworm (T. solium) which has the potential to lead to human neurocysticercosis, with serious, debilitating and possibly fatal consequences. As these tapeworms pose a dramatic health risk, infected carcasses discovered during inspection at the slaughter house are condemned as a matter of public policy.
Present inspection of livestock or meat for cysticercosis infection requires dissection and visual examination of specimens, a tedious, time-consuming and labor-intensive practice. The direct meat inspection is done using organoleptic means based on prescribed procedures designed to optimize detection of lesions. These postmortem examinations for cysts are extremely insensitive. It is estimated that organoleptic inspections for metacestode cysts performed at the time of slaughter misdiagnose in excess of 25% of infected animals as falsely negative. The detection of Taenia saginata and Taenia solium, the causative agents of cysticercosis, continue to pose large economic losses and serious health concerns in a number of countries, including the United States.
As yet, serological diagnostic tests for screening livestock have not been available due to the lack of specific and sensitive antigen reagents. Previous efforts to develop specific and sensitive antigens have not met with success. Given the moderate level of host antibody responses to cestode infections and the low numbers of cysts often associated with naturally-infected animals it was necessary to develop a highly sensitive and specific test.
Antigen reagents which have been evaluated by the ELISA method for the detection of T. saginata antibodies include an adult cestode extract which is reported to have strong cross-reactivity with antibodies to Fasciola hepatica, a common trematode parasite of cattle Craig et al., Zeitschrift fur Parasitenkunde 61:287-297 (1980)!.
A de-lipidized saline extract of the larval stage of the heterologous cestode Taenia crassiceps was evaluated as an immunodiagnostic antigen and though it exhibits a high degree of specificity for T. saginata antibodies (4.3% false-positive reaction) it has an unacceptably low degree of sensitivity for antibodies in both naturally (62% false-negative reaction) and experimentally infected animals Geerts et al, Res. Vet. Sci., 30:288-293 (1981)!.
A heterologous antigenic fraction purified from Taenia hydatigena, termed ThFAS, demonstrated reliable detection of T. saginata antibodies using the enzyme linked-immunosorbent assay (ELISA) where the specific immunodiagnostic reactivity of this fraction was associated with a 10 kDa protein. See Rhoads, M. et al., J. Parasitol. 71:779-787 (1985); Kamanga-Sollo, E. et al., Proc. Am. Assoc. Vet. Parasitol. 31:31 (1986); and Rhoads, M. et al., Vet. Arch., 57(3)143-150 (1987). As ThFAS is derived from naturally-occurring sheep cestodes, limitations have been encountered in obtaining the antigen in sufficient quantities and purity for use in a viable commercial test. Obtaining the homologous antigen from T. saginata cysts presents a problem since the muscle cysts and hence the mRNA from T. saginata, is difficult to purify free of host material. Consequently, the identification of an alternative source is necessary.
Efforts to develop an assay for the diagnosis of cysticercosis in humans has also been less than completely successful. Currently, the ELISA method shows the highest accuracy of immunodiagnosis for human cysticercosis. Using either a crude extract of swine cysticerci as an antigen or a purified fraction thereof (antigen B), antibodies were detected by ELISA in sera or cerebrospinal fluid (CSF) in only 70 to 80% of the clinically diagnosed cases Diwan et al., Am. J. Trop. Med. Hyg. 31:364-369 (1982); and Espinoza et al., Cysticercosis. Present State of Knowledge and Perspectives, 163-170, Academic Press, New York (1982)!. In addition, non-specificity has been demonstrated using this method (Coker-Vann et al., Trans. Roy. Soc. Med. Hyg. 78:492-496 (1984)). However, false-positive reactions can be eliminated by combining ELISA with an immunoblotting technique Gottstein et al, Am. J. Trop. Med. Hyg. 35:308-313 (1986) and Trop Med. Parasitol. 38:299-303 (1987)!. Recently, an ELISA using any one of three purified proteins isolated from the scolex of T. solium metacestodes by monoclonal antibody-immunoaffinity chromatography was able to detect 100% of the patients with cysticercosis and showed no false-positives (Nascimento et al 1987). In all of these human cysticercosis assays, the antigenic reagent was obtained from the homologous cestode, T. solium. Furthermore, to achieve accurate and reliable assays it is necessary to use additional expensive and time-consuming techniques.
These techniques would be completely unacceptable as a screening tool for the inspection of livestock or meat. An acceptable screening method must be capable of diagnosing cysticercosis in livestock or meat with a high degree of both sensitivity and specificity while remaining cost effective. Such an immunoassay screening method requires an appropriately reactive antigen. Since availability of antigen is a major limiting factor in diagnostic test development and production, reliable sources of antigen must be secured and the antigen must be available in quantities sufficient to support field trials and large scale test implementation.